Detection of Anaplasma phagocytophilum in wild boar in Slovenia

Abstract

Human granulocytic anaplasmosis (HGA) comprises a group of emerging tick-borne infectious diseases and it is caused by the intracellular bacteria, Anaplasma phagocytophilum. Infections with Anaplasma spp. have been described in humans and animals. Many studies have been performed to elucidate the natural cycle of A. phagocytophilum, but a competent reservoir host has not been clearly established in Europe [1]. Roe and red deer represent a very important host for the adult tick I. ricinus, a vector of A. phagocytophilum, and could therefore also serve as a natural reservoir for A. phagocytophilum in enzootic transmission of this bacterium [1]. Two genetic lineages of groESL operon were described among isolates of A. phagocytophilum from deer in Slovenia. However, both genetic lineages differed from a single variant, found in all Slovenian HGA patients [2]. PCR screening of different wild animals from Austria and the Czech Republic showed that wild boar (Sus scrofa) were infected with A. phagocytophilum [3]. Nucleotide sequences of groESL operon of A. phagocytophilum obtained from these infected animals were identical to the sequences from Slovenian patients [3]. Interestingly, a German horse with equine granulocytic ehrlichiosis was also infected with an identical variant of A. phagocytophilum to that detected in Slovenian patients [4]. In the years 2002 and 2007, 248 wild boars (Sus scrofa) from different locations in Slovenia were shot by professional hunters. Blood and spleen samples collected by hunters were analysed for infection with Anaplasma species in our laboratory. DNA was extracted from 135 spleen (year 2002) and 113 blood (year 2007) samples as described previously [1]. For the initial screening of all samples a segment of the 16S rDNA of A. phagocytophilum was used [1]. All positive samples were additionally amplified with a nested PCR targeting groESL operon of variants of A. phagocytophilum [1]. The gene for 16S rDNA is very conservative, therefore only the amplicons of the groESL operon were further analysed by sequencing. To obtain the genetic variation of A. phagocytophilum, all amplicons of groESL operon were further sequenced on both strands and analysed by using TREECON software. A phylogenetic tree was constructed with the neighbour-joining method. Support for the tree nodes was calculated with 1000 bootstrap replicates. Of 135 spleen samples (2002) and 113 blood (2007) samples, six (4.4%) and four (3.5%) tested positive for the presence of Anaplasmae spp. DNA, respectively. The groESL gene was subsequently amplified from all positive samples. The homology search and the alignment of groESL sequences showed 100% identity with A. phagocytophilum from a human patient (acc. no. AF033101) and from the Ixodes ricinus tick (acc. no. EU246961) from Slovenia (Fig. 1). Genetic sequences from wild boar were also 100% identical among each other. Our study confirms that wild boar is naturally infected with A. phagocytophilum. The Anaplasmae spp. DNA was detected in 10 of 248 (4.0%) samples, collected from different locations in Slovenia in two different years. Anaplasma phagocytophilum was previously detected in wild boar samples. Interestingly, the wild boar were hunted in a different geographical area (Czech Republic) but the results were in accordance with our findings [3]. Scandinavian groESL sequences from dogs and horses differ from Slovenian in six nucleotides, but are identical among each other. These differences might suggest geographical variability of A. phagocytophilum and contribute Corresponding author and reprint requests: Tatjana Avsic Županc, Institute of Microbiology and Immunology, Faculty of Medicine, Zaloska 4, SI–1000 Ljubljana, Slovenia E-mail: tatjana.avsic@mf.uni-lj.si